molecular responses of european flounder (platichthys flesus) chronically exposed to contaminated...

Chemosphere xxx (2014) xxx–xxx

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Molecular responses of European flounder (Platichthys flesus) chronicallyexposed to contaminated estuarine sediments

http://dx.doi.org/10.1016/j.chemosphere.2014.01.0280045-6535/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +44 121 4143393; fax: +44 121 4145925.E-mail address: [email protected] (T.D. Williams).

1 Present address: Yantai Institute of Coastal Zone Research, Chinese Academy ofSciences, Yantai 264003, PR China.

2 Present address: Glasgow International College, Glasgow University, Glasgow G116NU, UK.

Please cite this article in press as: Williams, T.D., et al. Molecular responses of European flounder (Platichthys flesus) chronically exposed to contamestuarine sediments. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.028

Tim D. Williams a,⇑, Ian M. Davies b, Huifeng Wu a,1, Amer M. Diab c,2, Lynda Webster b, Mark R. Viant a,J. Kevin Chipman a, Michael J. Leaver c, Stephen G. George c, Colin F. Moffat b, Craig D. Robinson b

a School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UKb Marine Scotland Science, Marine Laboratory, 375 Victoria Rd., Aberdeen AB11 9DB, UKc Institute of Aquaculture, The University of Stirling, Stirling FK9 4LA, UK

h i g h l i g h t s

� Flounders were exposed to contaminated sediments in mesocosms for 7 months.� Little differential accumulation of contaminants was found by chemical analysis.� 1-Hydroxypyrene and DNA damage increased in exposed fish.� Metabolic and transcriptional changes were detected in fish livers.� Molecular alterations were consistent with chronic PAHs exposure.

a r t i c l e i n f o

Article history:Received 30 September 2013Received in revised form 13 January 2014Accepted 25 January 2014Available online xxxx

Keywords:SedimentsFlounderToxicogenomicsMicroarrayMetabolomicsGenotoxicity

a b s t r a c t

Molecular responses to acute toxicant exposure can be effective biomarkers, however responses tochronic exposure are less well characterised. The aim of this study was to determine chronic molecularresponses to environmental mixtures in a controlled laboratory setting, free from the additional variabil-ity encountered with environmental sampling of wild organisms. Flounder fish were exposed in meso-cosms for seven months to a contaminated estuarine sediment made by mixing material from theForth (high organics) and Tyne (high metals and tributyltin) estuaries (FT) or a reference sediment fromthe Ythan estuary (Y). Chemical analyses demonstrated that FT sediment contained significantly higherconcentrations of key environmental pollutants (including polycyclic aromatic hydrocarbons (PAHs),chlorinated biphenyls and heavy metals) than Y sediment, but that chronically exposed flounder showeda lack of differential accumulation of contaminants, including heavy metals. Biliary 1-hydroxypyreneconcentration and erythrocyte DNA damage increased in FT-exposed fish. Transcriptomic and 1H NMRmetabolomic analyses of liver tissues detected small but statistically significant alterations between fishexposed to different sediments. These highlighted perturbance of immune response and apoptotic path-ways, but there was a lack of response from traditional biomarker genes. Gene-chemical associationannotation enrichment analyses suggested that polycyclic aromatic hydrocarbons were a major classof toxicants affecting the molecular responses of the exposed fish. This demonstrated that molecularresponses of sentinel organisms can be detected after chronic mixed toxicant exposure and that thesecan be informative of key components of the mixture.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The majority of terrestrial and aquatic toxicology is conductedusing acute exposures to individual chemicals, usually at concen-trations higher than those detected in the environment. In con-trast, wild animals are typically chronically exposed to complexmixtures of chemical contaminants where each constituent is pres-ent at a relatively low concentration. Acute toxicology studies are

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http://dx.doi.org/10.1016/j.chemosphere.2014.01.028

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2 T.D. Williams et al. / Chemosphere xxx (2014) xxx–xxx

useful for determining toxicant modes of action, but relating theseresults to field studies is not straightforward, due to the differencesoutlined above and the non-pollutant variation inevitably encoun-tered in the environment. Certain biomarker genes, such as cyto-chrome P450 1A (CYP1A) and vitellogenins (VTG), have beenfound to be useful in revealing the classes of contaminants towhich wild animals have been exposed (van der Oost et al.,2003). In aquatic research, transcriptomics techniques have beenapplied both to acute toxicological studies (Van Aggelen et al.,2010) and, to a lesser extent, to field studies (Williams et al.,2003, 2011; Meyer et al., 2005; Falciani et al., 2008; Sellin Jeffrieset al., 2012; Asker et al., 2013). The European flounder fish (Platich-thys flesus) is a species employed for biomonitoring in North-Wes-tern European coastal areas (Kirby et al., 2004), where it is exposedto marine and estuarine sediments due to its demersal habitat.Using flounder we have demonstrated several important conceptswhich strongly support the use of omics based measurements inthe analysis of chemical exposures. Flounders sampled from differ-ently-contaminated environments display different gene expres-sion profiles (Williams et al., 2003). Molecular responses toindividual toxicants provide information on their mechanisms ofaction (Sheader et al., 2006; Williams et al., 2006, 2007) and differ-ent toxicants result in different hepatic gene expression profiles(Williams et al., 2008). Statistical models based on gene expressionresponses to single chemical exposures can discriminate betweenfish sampled from differently contaminated sites (Falciani et al.,2008) and network integration of multiple biomarkers, transcri-ptomics and metabolomics can partially predict both exposureand effects of aquatic pollutants in field-sampled animals(Williams et al., 2011).

In a study described previously (Leaver et al., 2010) we reportedthat hepatocytes isolated from flounders that had been exposed inmesocosms for 7 months to relatively clean, or multiply-polluted,sediments did show differences in gene expression. However thesedifferences were indicative of chronic inflammation, immune re-sponse and apoptosis, rather than corresponding to the classic bio-markers of environmental pollution (e.g. CYP1A, VTG). Subsequentacute treatment of the hepatocytes with model toxicants benzo(a)-pyrene, copper or estradiol did indeed elicit the expected bio-marker gene expression responses (Leaver et al., 2010), and thesewere modulated by the different prior chronic exposure conditions.Here, we instead focus on liver tissue from the individual fishesthat were maintained in the mesocosms, rather than pooled hepa-tocytes. We aimed to determine if the different sediment typeselicited different transcriptional and metabolic responses fromthe flounders, whether there was evidence of DNA damage andwhether there was accumulation of contaminants and their metab-olites in flounder tissue and bile. This would not only allow furthervalidation of the results from the previous hepatocyte study, butalso contribute to understanding the differences between chronicand acute chemical exposures.

2. Materials and methods

2.1. Experimental animals and mesocosms

Details of experimental animals and the mesocosms employedwere described previously by Robinson et al. (2009) and Leaveret al. (2010). Briefly, four 1.5 m tanks were established, containing4–5 cm depth of test sediment, supplied with �100 L h�1 carbon-filtered seawater. Two tanks contained sediment from the Ythanestuary (Y) (North East Scotland, UK) and two tanks contained amixture of sediments (FT) from the Forth estuary (Grangemouth,UK) and the Tyne estuary (Riverside Quay, Jarrow, UK). The Ythan

Please cite this article in press as: Williams, T.D., et al. Molecular responses ofestuarine sediments. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemos

sediment was chosen as a control as discrimination betweensediments containing low levels of pollutants and those containinghigh levels is key in environmental monitoring. The Forth and Tynesediments were mixed to include high levels of both metal and or-ganic pollutants. Sediment chemical analyses have been describedpreviously (Leaver et al., 2010). All mesocosms were stocked withbenthic invertebrates sampled from the Ythan and from the Firthof Tay (Balmerino, NW Scotland, UK). Juvenile flounders (Platich-thys flesus) were caught from the Ythan estuary and 60 fish(3.1 ± 1.1 g, 56 ± 6 mm) were added to each mesocosm. Fish wereadditionally fed with mysid shrimp and commercial pellets (Bio-Optimal START 1.5 mm; Biomar, Grangemouth, UK). Tanks wereregularly inspected and any dead fish removed. After 7 monthsthe majority of the fish were sampled for transcriptomic and met-abolomic analyses, while the remaining fish were maintained inthe mesocosms for a further 7 months before sampling. A numberof morphometric parameters were measured to assess the generalhealth and stage of sexual development, including the gonadoso-matic index (GSI, gonad wt/body wt � 100), the hepatosomatic in-dex (HSI, liver wt/body wt � 100), and the condition factor (bodywt/(length)3 � 100). Blood was taken from a subset of individualsinto heparinised micro-haematocrit tubes and immediately trans-ported on ice. For gene expression and metabolomic analysis, sam-ples of liver and muscle tissue and bile were snap frozen and storedat �80 �C; tissue samples for chemical analysis were frozen at�20 �C.

2.2. Tissue chemistry

Chemical analyses were carried out for trace metals and for bil-iary 1-hydroxypyrene (a PAHs metabolite) on flounder muscle tis-sue at the 7 month sampling point (n = 55 per sediment). At the14 month sampling point, trace metals were analysed in pooledmuscle and in pooled liver samples (n = 2–4) and chlorinatedbiphenyls were also measured in pooled muscle and pooled livertissue. Metal and chlorinated biphenyl concentrations were deter-mined as shown previously (Robinson et al., 2009; Leaver et al.,2010). The concentration of conjugated 1-hydroxypyrene in floun-der bile was determined following the fixed wavelength fluores-cence method (Aas et al., 2000) as recommended by theInternational Council for the Exploration of the Sea (ICES) for useas a routine screening tool to assess recent PAHs exposure (Arieseet al., 2005). Bile samples were initially diluted 1600-fold in meth-anol and the concentration of 1-hydroxypyrene determined bycomparison with reference calibration standards.

2.3. Comet assay

The alkaline single cell gel electrophoresis Comet assay allowsassessment of DNA damage by detecting migration of DNA fromthe immobilised nucleus. The method described by Emmanouilet al. (2006) was employed. Briefly, fresh blood cells (mainly eryth-rocytes) were immobilised in agarose, layered onto microscopeslides, lysed, subjected to electrophoresis (25 V, 300 mA, 20 min)and stained with ethidium bromide. The Comets were examinedusing a fluorescent microscope (�20 magnification, Zeiss Axiovert10 inverted fluorescent microscope; Carl-Zeiss, Jena, Germany).The cells were scored using an image analysis package (Comet3.0 Europe Kinetic Imaging, Liverpool, UK). 100 cells were scoredper individual animal (Tice et al., 2000). Median percentage ofDNA in tail (% tail DNA) was compared for 25 fish from Ythan mes-ocosms and 25 fish from Forth/Tyne mesocosms. Data were ana-lysed in SPSS (version 20; IBM), shown not to be normally

European flounder (Platichthys flesus) chronically exposed to contaminatedphere.2014.01.028


T.D. Williams et al. / Chemosphere xxx (2014) xxx–xxx 3

distributed by the Shapiro–Wilks test and therefore were com-pared using the Mann–Whitney U test.

2.4. Microarray experiments

Samples of livers from flounders exposed to Ythan sediment (12male, 16 female) and Forth/Tyne sediment (15 male, 22 female)were analysed by transcriptomics and metabolomics. Liver tissuesfrom individual flounders were homogenised in 8 ml g�1 (v/w wetweight) methanol and 2.5 ml g�1 (v/w) water using a bead-basedhomogenizer (Precellys 24; Stretton Scientific, Stretton, UK) (Wuet al., 2008). Aliquots were then taken for both metabolomicsand transcriptomics (Katsiadaki et al., 2010). The GENIPOL Euro-pean flounder microarray (Williams et al., 2006) was used fordetermination of differential gene expression. This microarray con-sisted of 13 824 flounder clones and controls spotted in duplicateonto Corning UltraGAPS slides (Corning Inc., Corning, NY, USA)with an MGII robot (Biorobotics, Cambridge, UK) at BirminghamUniversity Functional Genomics Laboratory, representing approxi-mately 3336 unique genes. We have previously shown details ofclones incorporated into the microarray (Williams et al., 2003,2006, 2007; Diab et al., 2008). Microarray experiments were car-ried out as described previously (Diab et al., 2008). Briefly, totalRNA samples were purified from liver tissue homogenates fromindividual fish by RNEasy (Qiagen, Crawley, Oxford), with DNAcontamination removed by DNA-free (Ambion, Austin, TX, USA)followed by spectrophotometry for quality control. RNA sampleswere reverse transcribed to cDNA (Superscript II; Invitrogen, Pais-ley, UK) then labelled with Cy5-dCTP (GE Lifesciences, Amersham,UK) using Klenow polymerase (Invitrogen). Each array consisted ofCy5-labelled cDNA from one individual fish hybridised versus aCy3-labeled artificial reference sample (Diab et al., 2008). Hybridi-sations were carried out for 18 h, before stringent washing andscanning with an Axon 4000B scanner (Molecular Devices, Wok-ingham, UK). Data were captured using Genepix software (Molec-ular Devices), and each slide was checked in detail, with spotsshowing poor morphology or arrays showing experimental arte-facts being repeated. MIAME compliant data have been submittedto ArrayExpress and assigned the accession E-MTAB-1847. Thedata used in analyses consisted of local background-subtractedmedian intensities. Microarray data were quantile normalizedusing geWorkbench software version 2.0.1 (Floratos et al., 2010).Only data from spots designated as ‘present’ were used. Data forlow intensity, highly variable spots were removed.

2.5. Metabolomics

For metabolomics, liver homogenate aliquots were further ex-tracted using methanol/chloroform/water (2:2:1.8 final volumes)(Bligh and Dyer, 1959; Wu et al., 2008). One-dimensional 1HNMR spectroscopy was performed upon the hydrophilic fractionas previously described (Katsiadaki et al., 2010). Briefly, NMR spec-tra were measured at 500.11 MHz using an Avance DRX-500 spec-trometer and cryogenic probe (Bruker, Coventry, UK), with 200transients collected into 32 k data points. NMR data sets werezero-filled to 64 k points, exponential line-broadenings of 0.5 Hzwere applied before Fourier transformation, and spectra werephase and baseline corrected, then calibrated (TMSP, 0.0 ppm)using TopSpin software (version 1.3; Bruker). The subsequent pro-cessing and statistical analyses of the NMR data were similar tothose described in a previous study (Katsiadaki et al., 2010). Resid-ual water was removed, each spectrum was segmented into0.005 ppm bins, and the total area of each binned spectrum wasnormalized to unity so as to facilitate comparison between thesamples and subjected to a generalized log transformation.


2.6. Omics data analyses

Metabolomic and transcriptomic datasets were normalised, logtransformed and scaled, then combined for analysis. Lists of tran-scripts and metabolites that changed in abundance between Ythanand Tyne/Forth sediment-exposed fish were generated using TMEVsoftware (Saeed et al., 2006) using the Rank Products (RP) test,employing a multiple-test-corrected q-value cut-off of 0.05, calcu-lated by permutation. Data from male and female fish were consid-ered together as both sets of samples had similar sex ratios(Y = 43% male, TF = 41% male). Enrichment analyses were carriedout at the DAVID website (Huang et al., 2009a,b) for genes withidentifiable human orthologs, employing a false discovery rate(FDR) cut-off of 0.05 or 0.1. All detected identifiable genes wereused as a background set for comparison. Enrichment analysis ofgene-chemical annotation, derived from the Comparative Toxicol-ogy Database (CTD) (Davis et al., 2009) was performed using theEASE software package within TMEV using a FDR cut-off of 0.1(Williams et al., 2011). Briefly, for presumed orthologs of all genesdetected by microarray, chemical to gene expression relationshipsfor any species were downloaded from CTD, segregated into induc-tion and repression and used to annotate each gene with its ‘chem-ical inducers’ and ‘chemical repressors’. Gene-lists wereinterrogated for enrichment of annotation by EASE in comparisonwith all genes detected. False-discovery rates were multiplied ifthe same chemical matched to induced gene lists as an inducerand to repressed gene lists as a repressor.

3. Results

3.1. Chemical analyses and morphological parameters

Sediment chemistry data have been reported previously (Leaveret al., 2010). Briefly, chemical analyses showed that, in comparisonto Ythan sediment, Forth/Tyne sediments contained significantly(P < 0.05) higher concentrations of hexachlorobenzene (HCB), totalorganochlorine pesticides (OCP), chlorinated biphenyls (CB), naph-thalenes, total polycyclic aromatic hydrocarbons (PAHs), and B[a]Pequivalents of PAHs, tributyltin, dibutytin, As, Cr, Cu, Hg, Ni, Pb andZn. Dichlorodiphenyltrichloroethane (DDT) and its metaboliteswere not significantly different. Cd was detected in Forth/Tynebut was below the limit of detection in Ythan sediment. In contrast,the flounder muscle tissues showed no significant differences in As,Cd, Cr, Cu, Mn, Hg or Zn concentrations between Forth/Tyne andYthan sediment-exposed fish after 7 months or in liver after14 months. There was a significantly higher concentration of Pbin FT fish muscle after 7 months (FT = 0.038 ± 0.092 mg/kg;Y = 0.014 ± 0.016 mg/kg; P < 0.001). Chlorinated biphenyl concen-trations were no different in muscle but appeared elevated in liverafter 14 months, though not statistically significantly (sum of liverPCBs: FT = 31 lg/kg; Y = 19.8 lg/kg). Liver chlorinated biphenylconcentrations were an order of magnitude higher than thosefound in muscle (P < 1.4E�6). Biliary 1-hydroxypyrene appearedelevated in Forth/Tyne sediment exposed fish (1.7 ± 1.3 lg/ml)compared with Ythan sediment exposed fish (0.9 ± 0.5 lg/ml) after7 months (P = 0.065, Mann–Whitney U test). No measured mor-phological parameters significantly differed between sexes or sed-iment-exposed groups, apart from gonad weights and GSI thatwere higher for female fish than males.

3.2. Comet assay

The flounders exposed to Ythan sediments showed 3.8% meantail DNA while those exposed to Forth/Tyne sediments showed amean 7.8% tail DNA (Fig. 1A). These data were variable for both



Fig. 1. (A) Mean of median percent tail DNA for flounders exposed to Ythan or Forth/Tyne sediment as measured by Comet assay. Error bars show standard error of the mean,n = 25 fish, 100 cells per fish, star indicates Mann–Whitney U test P-value <0.05. (B) Fish categorized by mean of median percent tail DNA for flounders exposed to Ythan orForth/Tyne sediment as measured by Comet assay. Numbers of fish are shown with 0–5%, 5–10% and above 10% median tail DNA, n = 25 fish, 100 cells per fish, for both groups


groups (Fig. 1B) but were found to be statistically significant by theMann–Whitney U test with P-value of 0.015.

3.3. Transcriptional and metabolic changes

The changes in metabolite and transcript abundance betweenflounders exposed to the different sediments for 7 months werenot extensive. Indeed, hierarchical clustering and principal compo-nents analysis of the data set failed to identify any relationshipsbetween overall gene expression or metabolic profiles and sedi-ment type. Rank Products analysis identified 259 transcripts andmetabolites significantly (q < 0.05) altered in abundance in Forth/Tyne sediment exposed fish compared with Ythan sediment ex-posed fish in both males and females. Data are shown in full inSupplementary Table 1. Of the transcripts induced with FT sedi-ment, 51 were identified and 45 were not identified. Of those re-pressed with FT sediment, 76 transcripts were identified and 69were not identified (Table 1). Six metabolites significantly in-creased in concentration with FT sediment and 12 significantly de-creased, but none of these could be identified. A number ofmetabolites similarly expressed in both groups were identified;choline, glucose, glutamine, glycine, lactate, malonate, maltose ortaurine, N,N-dimethylglycine, o-phosphocholine, taurine, tyrosineand valine. The statistically significant changes in transcriptexpression and metabolite concentration were relatively modestin magnitude between FT and Y samples, with only 17% of thoseidentified exceeding a 1.5-fold change in males and 6% in females.

3.4. Annotation analyses

Gene Ontology annotation enrichment analysis, carried outusing DAVID, identified significant (FDR < 0.05) enrichment of theGO term ‘iron ion binding’ (GO:0005506) among transcriptsup-regulated with FT sediment and significant enrichment(FDR < 0.05) of the GO term ‘calcium ion binding’ (GO:0005509)among transcripts down-regulated with FT sediment, as well as a


less significant (FDR < 0.1) enrichment of ‘microsome’(GO:0005792).

Gene-chemical annotation enrichment analysis highlightedchemical pollutants whose associated gene expression changeswere consistent with those detected in response to TF versus Y sed-iment in this experiment (Table 2). These included fungicides (pro-cymidone, vinclozolin and trichostatin A), the polycyclic aromatichydrocarbon benzo(a)pyrene, a brominated flame retardant(BDE-47), a fluoro-surfactant (PFOS) and a model genotoxichepatocarcinogen (N-nitrosomorpholine). The highest number ofmatches between gene expression changes in these floundersand those in CTD were for benzo(a)pyrene (56 matches).

4. Discussion

Despite FT sediment containing significantly higher concentra-tions of a range of organic and inorganic contaminants than Y sed-iment, there was little evidence of differential accumulation ofthese contaminants in flounder tissue. It is therefore possible thatthe majority of contaminants assayed were not bioavailable to theflounders, despite their demersal foraging behaviour. The onlyexceptions were a higher, but variable, concentration of lead inFT fish muscle and an increase in biliary 1-hydroxypyrene. The bil-iary 1-hydroxypyrene elevation was consistent with exposure toand metabolism of pyrenes (Ariese et al., 2005), members of thegroup of polycyclic aromatic hydrocarbons (PAHs). PAHs arewell-known genotoxins, and may have contributed to the signifi-cantly elevated DNA damage detected in FT flounder blood cellsby the Comet assay. ICES recommend a Background AssessmentCriteria (BAC) value for the Comet assay of 5% DNA in tail to assesswhether exposure to genotoxins has occurred in fish (dab and cod;Davies et al., 2012); the values here of 3.8% in the Y fish and 7.8% inthe FT fish are consistent with the latter being exposed to genotox-ins and the former not being highly exposed. PAHs tend not toaccumulate chronically in fish tissue (Du-Lacoste et al., 2013)



Table 1Transcripts significantly changing (rank products q value <0.05) between Forth/Tynesediment-exposed and Ythan sediment-exposed flounders after 7 months.

Induced Repressed

Immune response Immune responseHAMP, ILDR1, ITGB2, KL, SERPINB4,

TP53INP2CD209, CD40, CCL8, C20ORF186, HP,IFI30, LEAP2, NCF2

Transcription TransportS100A1, TWISTNB, EGR1, MED19,

NR1D1, ZNF234AQP1, EEA1, SLC2A2, SLC25A12,SLC27A1, SNX12

Xenobiotic metabolism Protein DegradationADH6, GSTO1, GSTT1, MIOX SEC11A, PSMB1, PSMB7, USP14,

UBE2CBP, CBPB1, ELA1Translation TranscriptionEIF4BP3, MRPL14, NOCL3, RPLP1 C1ORF85, MLL4, SYNCRIP, TRIP13,

AP1GBP1, HNRPUL1Energy Xenobiotic metabolismCYTB, G6PC, PPP1CC, RENBP UGT1A1, ALDH, ALDH2, CMBL, CYP1AApoptosis ApoptosisDIABLO, NUPR1, PTRH2 HTRA2, CASP3, PTMA, TRIM35Signal transduction EnergyMBIP, RAB11A, PGRMC1 ND3, GCK, FLJ44606, IDH2Proteolysis Calcium bindingCPN1, UBE2V2, UBE2Q2 S100A14, CAL1, CALM3, CANXCytoskeleton CytoskeletonMPP1, TM4SF1, TUBA1A ACTN1, PSTPIP2, SDC2, MTSS1LAmino acid metabolism Lipid metabolismARG2, TDO2 APOB, PLA2G1B, PTGES3Cell cycle Amino acid metabolismZW10, INTS3 FAH, SEPHS2Nucleotide metabolism Cell cycleATIC RAN, GFI1BProtein modification TranslationNAGK GUF1, DENRTransport Signal transductionSLCO1C1 DDIT4L, RTN1Lipid metabolism Nucleotide metabolismCYP24A1 DPYD, HPRT1Oxygen Transport Cell adhesionHBE1 EMILIN1, NCAN

ChaperonesHSP90B1, HSP90AOthersHHATL, PRDX1, BLVRA, GIF

Table 2Chemical-transcript association enrichment analysis. FDR denotes false discoveryrate. Numbers of transcripts whose regulation matched those found in CTD are noted(induced and repressed).

Term FDR Induced Repressed

Procymidone 0.083 2 2Benzo(a)pyrene 0.087 28 28N-nitrosomorpholine 0.087 2 2Vinclozolin 0.093 6 4Trichostatin A 0.097 5 5Tetrabrominated diphenyl ether 47 0.099 3 2Perfluorooctane sulfonic acid 0.099 4 8


due to their metabolism by cytochromes P450 and subsequentconjugation and excretion via the bile.

Alterations in gene expression and metabolite concentrationswere detectable, but not of great magnitude. Again this was consis-tent with low bioavailability leading to modest hepatic exposure.While none of the metabolites that altered in concentration wereidentifiable, the detection of metabolic change in these chronicallyexposed animals adds weight to the finding that chronic exposuredoes elicit functional phenotypic alterations in the long term.Although the magnitude of gene expression changes was small, itwas evident that there were similarities with the responses of iso-lated hepatocytes and qPCR data (CYP1A, hepcidin, DIABLO) onadditional fish from the same mesocosms described by Leaver


et al. (2010).Transcripts differentially expressed between TF andY flounders fell into a number of functional categories. The cate-gory with the greatest number of differentially expressed tran-scripts was ‘immune response’ (Table 1), as was found inhepatocytes (Leaver et al., 2010), with a similar induction of hepci-din, encoding an antibacterial peptide. Additionally DIABLOexpression was induced in FT fish similarly to responses of isolatedhepatocytes. This transcript has recently emerged as a possible bio-marker of chronic chemical contamination (Zacchino et al., 2012),with the DIABLO protein playing a key role in promoting apoptosis.Therefore the transcriptomic responses detected in fish livers werebroadly consistent with those found in isolated hepatocytes, indic-ative of alteration of immune response and apoptosis. Indeed,these classes of responses have been detected in other fish speciesdirectly sampled from polluted environments (Asker et al., 2013).

Similarly to Leaver et al. (2010) we found that typical biomarkergenes were not induced in FT fish. Indeed, the transcription ofCYP1A was repressed, as was that of UDP-glucuronosyltransferase(UGT), despite the evidence for increased biliary PAHs metabolites.As many PAHs are effective inducers of CYP1A expression andactivity via activation of the aromatic hydrocarbon receptor, thiswas unexpected. There are a number of possible reasons for a de-cline in CYP1A transcription. As outlined previously there mayhave been insufficient exposure or a lack of responsiveness afterlong term exposure, with transcription returning to equilibrium(Reynolds et al., 2003; Leaver et al., 2010). If so, this demonstratesone of the main differences between acute and chronic responses.Alternatively, there may be mixture effects interfering with CYP1Atranscription, such as the repression of PAHs-induced CYP1A tran-scription in flounder when co-exposed with heavy metals such ascadmium (Lewis et al., 2006). This is not necessarily an effect med-iated solely by heavy metals, as estrogens (Maradonna et al., 2004)and polybrominated diphenyl ethers (Wahl et al., 2010), amongstmany other compounds, have been found to repress CYP1A in fish.It is possible that such repression is mediated by oxidative stress(Morel and Barouki, 1998). Both time-dependent and mixture-dependent factors may contribute to CYP1A repression in thisscenario. Although transcription of CYP1A, UGT and aldehydedehydrogenases ALDH and ALDH2 decreased in FT fish, there wasinduction of glutathione-S-transferases (GST) of the omega andtheta classes and of alcohol dehydrogenase. While GSTs are keycomponents of phase II metabolism of xenobiotics such as PAHs,GST induction is also associated with oxidative stress (Leaveret al., 1997), and this is not inconsistent with chronic inflammationand immune response.

While the transcriptional changes detected were modest andinitially appeared non-specific, interrogation of previous literaturevia CTD gene-chemical annotation enrichment analysis did identifysignificant (FDR < 0.1) similarities between gene expression re-sponses recorded in response to individual chemical exposuresand the gene expression responses found to this chronic complexmixture exposure (Table 2). Most notably, there was significantsimilarity between the genes differentially expressed between FTand Y fish and the genes previously found to respond to benzo(a)-pyrene treatment. This implies that, while the individual bio-marker approach can be very useful, especially in acute studies, amulti-biomarker readout is more effective at identifying stressorsaffecting chronically exposed animals. Additional similaritiespredicted included fungicides (procymidone, vinclozolin andtrichostatin A), a brominated flame retardant (BDE-47), afluoro-surfactant (perfluorooctane sulfonic acid, PFOS) and a modelgenotoxic hepatocarcinogen (N-nitrosomorpholine). BDE-47 wasindeed detected in the FT sediment (Robinson et al., 2009) andthe presence of fungicides and PFOS would not be unexpected incontaminated estuarine sediment. N-nitrosomorpholine is unlikelyto occur in detectable concentrations in the environment, but




effectively illustrates detection of the DNA damage that was alsoconfirmed by Comet assay.

5. Conclusions

Chemical analyses of flounder chronically exposed to morehighly contaminated Tyne/Forth estuarine sediment demonstrateda lack of contaminant accumulation in fish tissues. Transcriptomicand metabolomic analyses of liver tissue detected small but statis-tically significant alterations between the groups of fish exposed todifferent sediments. These broadly confirmed the results of a pre-vious study on isolated hepatocytes, highlighting alterations in im-mune response and apoptosis, but a lack of response fromtraditional biomarker genes. Together with analyses of fluorescentbile metabolites and DNA damage, the data were consistent withuptake, metabolism and excretion of genotoxic polycyclic aromatichydrocarbons. Gene-chemical association annotation analyses alsosuggested that PAHs were a major class of toxicants affecting themolecular responses of contaminant exposed fish. The study high-lighted the importance of bioavailability and the benefits of a mul-ti-biomarker approach in determining responses to chronicchemical mixture exposure.

Acknowledgements

This work was funded by UK NERC Post-genomic and Proteomicprogramme Grant NE/C507661/1, UK BBSRC and EU GENIPOL pro-gramme Grant EKV-2001-0057.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2014.01.028.

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